The purpose of this project is to study the action of diphtheria toxin at the cellular and molecular level, and to compare and contrast the actin of Pseudomonas exotoxin A, as this toxin has the same cellular target but a different mechanism of entry into the cell. Our major objectives include: 1) Determination of the presence and activity of toxin-specific receptor sites and determination of the process by which diphteria toxin enters cells and its active A fragment reaches the cytosol. 2) Determination of the cellular and molecular basis for the resistance to diphtheria toxin found in some cells and animal species. 3) Elucidation of steps which lead to inhibition of protein synthesis in intoxicated cells. 4) Elucidation of steps in the biosynthesis of diphthamide and determination of its biological role. 5) Extension of our study to include the interaction of toxin-sensitive and toxin-resistant cells with other biologically active agents, including toxins, viruses, hormones, and transport proteins. Investigations will be conducted using cultured mammalian cells and cell-free systems. In our laboratory we have isolated a diverse group of toxin-resistant strains of human and non-human cells which are blocked at different steps in the intoxication process, some at the level of toxin entry and some at the target enzyme elongation factor 2. Utilizing a combination of biochemical and genetic techniques, and techniques of ultrastructural analysis, we have a unique opportunity to investigate not only sensitivity and resistance to microbial toxins, but also regulation of gene expression and cellular function.